MPRAsnakeflow experiment QC report

WarningLow correlation warning!

At least one (not all) Spearman correlations across replicates is low using a barcode threshold per oligo of 10! Spearman correlation of DNA is 0.95, RNA is 0.60 and DNA/RNA ratio is 0.65. Minimum allowed values are 0.85, 0.95, and 0.75.

Overall quality metrics

Table explanation
  • median rna read count: Median of RNA read count for oligos that passed filtering to determine sufficient coverage in terms of read count. Value is the median of all replicates.
  • median barcodes passing filtering: Median number of barcodes across tested sequences that passed filtering to determine if there was sufficient barcode to oligo coverage. Value is the median of all replicates.
  • pearson correlation: The correlation of log2 RNA/DNA ratios across tested sequences as a measure of replicable activity signal. Value is the median of replicate comparisons using only oligos with >= 10 barcodes.
  • fraction oligos passing: Fraction of tested sequences that passed filtering of the mappable sequences to determine if the designed library was sufficiently recovered. Value is the median of all replicates and using only oligos with >= 10 barcodes.
median barcodes passing filtering median rna read count pearson correlation fraction oligos passing
67 641 0.85 0.92

DNA over RNA counts

Plotting normalized counts of DNA vs RNA (median across replicates). Only oligos within all replicates are shown. We should see a variation within the RNA count data (along the y axis). If count data between RNA and DNA is highly correlated (e.g. follows the identity line) there is no variation between designed oligos. This is an indication that RNA is inflated with DNA and the DNA digestion before creating cDNA did not work as expected.

Oligo correlation

Oligo correlation plots of DNA, RNA and DNA/RNA ratios across replicates. First tab shows plots using (in average) 54114 oligos with a minimum number of 10 barcodes. Second tab shows all 58440 oligos that have assigned barcodes.

Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
A549 1 2 54791 54705 54328 0.96 0.63 0.67 0.96 0.82 0.84
A549 1 3 54791 54775 54367 0.96 0.64 0.67 0.96 0.83 0.85
A549 1 4 54791 54339 54066 0.96 0.62 0.67 0.96 0.82 0.84
A549 1 5 54791 54333 54057 0.96 0.60 0.65 0.95 0.81 0.84
A549 2 3 54705 54775 54305 0.96 0.66 0.71 0.96 0.86 0.88
A549 2 4 54705 54339 54045 0.96 0.64 0.70 0.96 0.84 0.86
A549 2 5 54705 54333 54026 0.96 0.63 0.70 0.95 0.84 0.87
A549 3 4 54775 54339 54054 0.96 0.65 0.71 0.96 0.85 0.87
A549 3 5 54775 54333 54061 0.96 0.64 0.70 0.96 0.86 0.87
A549 4 5 54339 54333 53831 0.95 0.61 0.70 0.95 0.84 0.86
Condition A B #Oligos A #Oligos B #Oligos Joined DNA spearman RNA spearman Ratio spearman DNA log2 pearson RNA log2 pearson Ratio log2 pearson
A549 1 2 58718 58727 58502 0.95 0.60 0.63 0.91 0.73 0.73
A549 1 3 58718 58716 58480 0.95 0.61 0.64 0.91 0.74 0.74
A549 1 4 58718 58649 58423 0.95 0.58 0.64 0.90 0.71 0.73
A549 1 5 58718 58638 58413 0.95 0.57 0.62 0.90 0.72 0.73
A549 2 3 58727 58716 58497 0.95 0.63 0.67 0.91 0.77 0.77
A549 2 4 58727 58649 58436 0.95 0.61 0.66 0.90 0.74 0.75
A549 2 5 58727 58638 58433 0.95 0.60 0.66 0.90 0.75 0.76
A549 3 4 58716 58649 58426 0.95 0.61 0.67 0.90 0.75 0.76
A549 3 5 58716 58638 58421 0.95 0.61 0.66 0.90 0.77 0.77
A549 4 5 58649 58638 58378 0.94 0.58 0.66 0.90 0.74 0.76

Experiment statistic

The total number of oligos in this experiment is 59308 (defined by the assignment) with 10381929 associated barcodes.

In average across replicates we see 58690 from 4912794 average barcodes in the count data and around 1166296 barcodes where not in the assignment.

condition replicate oligos dna/rna matched barcodes unknown barcodes dna/rna % matched barcodes total dna counts total rna counts avg dna counts per bc avg rna counts per bc barcode outlier removed avg dna/rna barcodes per oligo
A549 1 58718 5078773 1200052 80.89 201932147 92956866 32.16 14.80 0 86.49
A549 2 58727 5000998 1194821 80.72 199108959 100483452 32.14 16.22 0 85.16
A549 3 58716 5121001 1231584 80.61 202834242 83176133 31.93 13.09 0 87.22
A549 4 58649 4666533 1093679 81.01 188881416 77523105 32.79 13.46 0 79.57
A549 5 58638 4696666 1111345 80.87 189555867 75199143 32.64 12.95 0 80.10
Experiment Barcodes Counts Average counts Assigned barcodes Assigned counts Average assigned counts Fraction assigned barcodes Fraction assigned counts
A549.1.DNA 15667758 299222797 19.10 9522795 251662451 26.43 0.61 0.84
A549.2.DNA 15667758 299222797 19.10 9522795 251662451 26.43 0.61 0.84
A549.3.DNA 15667758 299222797 19.10 9522795 251662451 26.43 0.61 0.84
A549.4.DNA 15667758 299222797 19.10 9522795 251662451 26.43 0.61 0.84
A549.5.DNA 15667758 299222797 19.10 9522795 251662451 26.43 0.61 0.84
A549.1.RNA 7637312 95973168 12.57 5145721 79451916 15.44 0.67 0.83
A549.2.RNA 7707693 103808893 13.47 5067519 85874822 16.95 0.66 0.83
A549.3.RNA 7658028 85918859 11.22 5190860 71060361 13.69 0.68 0.83
A549.4.RNA 6935335 80063496 11.54 4726069 66157073 14.00 0.68 0.83
A549.5.RNA 6998535 77706392 11.10 4757847 64309380 13.52 0.68 0.83

Histograms barcodes per oligo, counts per barcode

Histogramm of number of barcodes per oligo and the number of counts per barcode devidied by DNA and RNA. Median is red, mean is blue.

Activity

Violin and box plots of the log2 fold change of all oligos by the assay. Grouped by labels if set, otherwise NA. First tab shows plots using (in average) 54114 oligos with a minimum number of 10 barcodes. Second tab shows all 58440 oligos that have assigned barcodes.